I tried with 50% FBS and 10% DMSO; 10% FBS and 5% DMSO; 5% FBS and 10% DMSO. But when recultured, in all the cases, around 30% of cells become dead.
The most gentle freezing media is 90% FBS. Some people use media. SOme people use media with 20% serum. Its almost always 10% DMSO. Look at the ATCC techniques carefully. Make sure your cell pellet is knocked loose, make sure it is chilled on ice for at least 20 minutes. make sure your freezing media is cold too. Transfer to -80 overnight encolsed in a styrofoam tube rack or a "Mr Frosty" type device, then to liq N2. When thawing, swirl the forzen amterial by hand in a 37 C bath, then when a small ice pellet is still visible, slowly drop in cold media with no serum. then when the freezing tube is near full draw up the material, and slow add to a centrifuge tube. It is already at 10% serum (9 ml cold plain media plus your 1 ml of frozen cell material). Pellet and wash 2x, and you should be fine
¡¡ suerte !! (good luck!)
Hola,
I usually work with SH-SY5Y cultures and I cryopreserve them taking 900 microliters of medium with the cells (F12 medium/EMEM + glutamine (2 mM) + 1% of non-essential amino acids + 15% of fetal bovine serum) + 100 microliters DMSO.
Hope it works in your hands!
Hello, Dr. Jose A Morales-Garcia
Thank you so much for your kind information. I will try with this condition and hope there will be some better output for me.....
These freeze down just fine in 90% FBS, 10% DMSO. Check out the ATCC guide for freezing and thawing cells.
Hello, Dr. Rimas,
Thanks a lot for your suggestion.According to ATCC, they recommended to use 5% DMSO with GM. I also tried with this, but same things happened.
The most gentle freezing media is 90% FBS. Some people use media. SOme people use media with 20% serum. Its almost always 10% DMSO. Look at the ATCC techniques carefully. Make sure your cell pellet is knocked loose, make sure it is chilled on ice for at least 20 minutes. make sure your freezing media is cold too. Transfer to -80 overnight encolsed in a styrofoam tube rack or a "Mr Frosty" type device, then to liq N2. When thawing, swirl the forzen amterial by hand in a 37 C bath, then when a small ice pellet is still visible, slowly drop in cold media with no serum. then when the freezing tube is near full draw up the material, and slow add to a centrifuge tube. It is already at 10% serum (9 ml cold plain media plus your 1 ml of frozen cell material). Pellet and wash 2x, and you should be fine
Dear Dr. Rimas,
So many thanks for your informative comments. I will try with your above mentioned conditions. I hope these will be better for cells and for me.
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